Effects of occupational therapy on improvements in the handwriting ability of the adult non-dominant hand: An exploratory randomised controlled trial.

INTRODUCTION: Occupational therapy often involves handwriting acquisition practices that include the non-dominant hand when improvements in the dominant hand function are not possible because of trauma or stroke. This study explored whether character tracing and using a pegboard can effectively improve the handwriting of the non-dominant hand. METHODS: A randomised controlled trial involving 60 healthy university students aged ≥18 years was conducted. Participants were randomly assigned to the writing group, peg group or control group. The character recognition rate was evaluated by computer software. Furthermore, character quality and writing speed were evaluated by humans using global legibility scales. Evaluations were performed before the intervention (baseline) and on days 5 and 10 of the intervention. Using the non-dominant hand, the writing group traced characters on paper with a ballpoint pen, and the peg group used a pegboard for 15 min/day for 10 days. RESULTS: Compared with the peg and control groups, the writing group showed significant improvements in the character recognition rate and global legibility scale score. However, the global legibility scale score did not improve to the same level as that achieved with the dominant hand. None of the evaluation scores of the peg group showed significant improvements compared with those of the control group. There were no significant differences in improvements in the writing speed of the writing and peg groups compared with the control group. CONCLUSION: Tracing characters can improve the handwriting ability of the non-dominant hand, but using a pegboard may be less effective. Future research is needed to examine how much practice is necessary to improve the handwriting ability of the non-dominant hand sufficiently.

Biotransformation and detoxification of tetrabromobisphenol A by white-rot fungus Phanerochaete sordida YK-624.

The bulky phenolic compound tetrabromobisphenol A (TBBPA) is a brominated flame retardant used in a wide range of products; however, it diffuses into the environment, and has been reported to have toxic effects. Although it is well-known that white-rot fungi degrade TBBPA through ligninolytic enzymes, no other metabolic enzymes have yet been identified, and the toxicity of the reaction products and their risks have not yet been examined. We found that the white-rot fungus Phanerochaete sordida YK-624 converted TBBPA to TBBPA-O-ß-D-glucopyranoside when grown under non-ligninolytic-enzyme-producing conditions. The metabolite showed less cytotoxicity and mitochondrial toxicity than TBBPA in neuroblastoma cells. From molecular biological and genetic engineering experiments, two P. sordida glycosyltransferases (PsGT1c and PsGT1e) that catalyze the glycosylation of TBBPA were newly identified; these enzymes showed dramatically different glycosylation activities for TBBPA and bisphenol A. The results of computational analyses indicated that the difference in substrate specificity is likely due to differences in the structure of the substrate-binding pocket. It appears that P. sordida YK-624 takes up TBBPA, and reduces its cytotoxicity via these glycosyltransferases.

The heterotrimeric G protein subunits Gα(q) and Gß(1) have lysophospholipase D activity.

In a previous study we purified a novel lysoPLD (lysophospholipase D) which converts LPC (lysophosphatidylcholine) into a bioactive phospholipid, LPA (lysophosphatidic acid), from the rat brain. In the present study, we identified the purified 42 and 35 kDa proteins as the heterotrimeric G protein subunits Gα(q) and Gß(1) respectively. When FLAG-tagged Gα(q) or Gß(1) was expressed in cells and purified, significant lysoPLD activity was observed in the microsomal fractions. Levels of the hydrolysed product choline increased over time, and the Mg(2+) dependency and substrate specificity of Gα(q) were similar to those of lysoPLD purified from the rat brain. Mutation of Gα(q) at amino acids Lys(52), Thr(186) or Asp(205), residues that are predicted to interact with nucleotide phosphates or catalytic Mg(2+), dramatically reduced lysoPLD activity. GTP does not compete with LPC for the lysoPLD activity, indicating that these substrate-binding sites are not identical. Whereas the enzyme activity of highly purified FLAG-tagged Gα(q) overexpressed in COS-7 cells was ~4 nmol/min per mg, the activity from Neuro2A cells was 137.4 nmol/min per mg. The calculated K(m) and V(max) values for lysoPAF (1-O-hexadecyl-sn-glycero-3-phosphocholine) obtained from Neuro2A cells were 21 µM and 0.16 µmol/min per mg respectively, similar to the enzyme purified from the rat brain. These results reveal a new function for Gα(q) and Gß(1) as an enzyme with lysoPLD activity. Tag-purified Gα(11) also exhibited a high lysoPLD activity, but Gα(i) and Gα(s) did not. The lysoPLD activity of the Gα subunit is strictly dependent on its subfamily and might be important for cellular responses. However, treatment of Hepa-1 cells with Gα(q) and Gα(11) siRNAs (small interfering RNAs) did not change lysoPLD activity in the microsomal fraction. Clarification of the physiological relevance of lysoPLD activity of these proteins will need further studies.

Difference between the Effects of Peripheral Sensory Nerve Electrical Stimulation on the Excitability of the Primary Motor Cortex: Examination of the Combinations of Stimulus Frequency and Duration.

Peripheral sensory nerve electrical stimulation (PES) excites the primary motor cortex and is expected to improve motor dysfunction post-stroke. However, previous studies have reported a variety of stimulus frequencies and stimulus duration settings, and the effects of these different combinations on primary motor cortex excitability are not clear. We aimed to clarify the effects of different combinations of stimulus frequency and stimulus duration of PES on the excitation of primary motor cortex. Twenty-one healthy individuals (aged > 18 years, right-handed, and without a history of neurological or orthopedic disorders) were included. Each participant experienced three different stimulation frequencies (1, 10 and 50 Hz) and durations (20, 40 and 60 min). Motor-evoked potentials (MEPs) were recorded pre- and post-PES. The outcome measure was the change in primary motor cortex excitability using the MEP ratio. We used a D-optimal design of experiments and response surface analysis to define the optimal combination within nine different settings inducing more satisfying responses. The combination of stimulation frequency and stimulation time that maximized the desirability value was 10 Hz and 40 min, respectively. The results of this study may provide fundamental data for more minimally invasive and effective implementation of PES in patients with stroke.

Purification and characterization of lysophospholipase D from rat brain.

A lysophospholipase D (lysoPLD) was purified to apparent homogeneity from rat brain nuclear fractions using 1-[(14)C]palmitoyl-glycerophosphorylcholine as a substrate. The abundance of autotaxin (ATX), a secretory lysoPLD, was also estimated for each fraction. The nuclear fraction had relatively high levels of lysoPLD activity but weak immunoreactivity with an anti-ATX antibody. LysoPLD activity was further purified 5550-fold by sequential chromatography. The final preparation migrated as a single band with a molecular weight of 35,000. Anti-ATX antibodies did not cross-react with the purified enzyme. Moreover, enzyme activity was highest at pH 7.0-7.5 and requires Mg(2+). The Km and Vmax values for 1-palmitoyl-glycerophosphorylcholine were 176 microM and 0.3 micromol/min/mg, respectively. The purified enzyme hydrolyzed saturated forms of LPC more robustly than unsaturated forms. The enzyme could hydrolyze platelet-activating factor (PAF) to the same extent as 16:0-LPC, and showed a higher activity toward lysoPAF (1-O-hexadecyl-2-lyso-glycerophosphorylcholine). These results suggested that the lysoPLD purified from rat brain nuclear fractions in this work is a novel enzyme that hydrolyzes lysoPAF, PAF, and LPC to liberate choline.

Influence of cytological stains on comparative genomic hybridization analysis for DNA extracted from cytological smears.

In the present study, we investigated the influence of cytological stains in analyzing DNA extracted from cytological slides by comparative genomic hybridization (CGH). Multiple imprint cytological slides were prepared for fresh-frozen breast cancer tissue samples and the slides were stained by three staining methods for each sample. Under microscopic observation, cancer cells were selectively microdissected from the slides and forwarded to DNA extraction, whole genome amplification, and CGH analysis. CGH was successfully performed for all methylgreen-stained and May-Grunwald-Giemsa (MGG)-stained cytological smear slides, but for two Papanicolaou (PAP)-stained slides. The number of chromosomal imbalances detected were 5-10 in methylgreen-stained slides and 5-9 in MGG-stained slides. The chromosomal imbalances resemble each other between methylgreen-stained and MGG-stained slides. The present study indicates that the MGG stain is preferred to the PAP stain for the purpose of cytogenetical analysis by CGH for DNA extracted from cytological smear slides.

Visual emotional context modulates brain potentials elicited by unattended tones.

To examine whether brain electrical responses to environmental stimuli were influenced by emotional contexts, event-related potentials (ERPs) elicited by nonstartle probe tones were recorded from 13 student volunteers while they were viewing emotionally positive, neutral, and negative slides of the International Affective Picture System. The auditory stimuli consisted of high-deviant (2000 Hz, p=.08), low-deviant (1050 Hz, p=.08), and standard (1000 Hz, p=.84) tones with a mean onset-to-onset interval of 600 ms. Participants were told to ignore the tones. High-deviant tones elicited a larger N1 (peaking around 100 ms) when participants were viewing negative slides than when viewing positive slides. The amplitude of the P2 elicited by standard tones (peaking around 170 ms) was smaller when participants were viewing positive slides than when viewing negative and neutral slides. The amplitude of the mismatch negativity (150-200 ms) tended to reduce during positive slide presentation, but this difference appeared to be due to reduction of the P2 elicited by standard tones. These findings suggest that visually induced emotional states have a sequential effect on auditory information processing, in that the influence of negative emotion appears at an earlier stage than that of positive emotion.

Amide compound synthesis by adenylation domain of bacillibactin synthetase.

The adenylation domain of nonribosomal peptide synthetase (NRPS) is responsible for the selective substrate recognition and its activation (as an acyl-O-AMP intermediate) during ATP consumption. DhbE, a stand-alone adenylation domain, acts on an aromatic acid, 2,3-dihydroxybenzoic acid (DHB). This activation is the initial step of the synthesis of bacillibactin that is a high-affinity small-molecule iron chelator also termed siderophore. Subsequently, the activated DHB is transferred and attached covalently to a peptidyl carrier protein domain via a thioester bond. Adenylation domains belong to the superfamily of adenylate-forming enzymes including acetyl-CoA synthetase, acyl-CoA synthetase and firefly luciferase. We previously reported a novel N-acylation reaction for an acyl-CoA synthetase (AcsA) that originally catalyzes the formation of a thioester bond between an acid and CoA, yielding acyl-CoA. This novel reaction was also confirmed for acetyl-CoA synthetase and firefly luciferase, but not yet for an adenylation domain. Here, we for the first time demonstrated the synthesis of N-acyl-L-cysteine by a stand-alone adenylation domain, DhbE. When DHB and L-cysteine were used as substrates of DhbE, N-DHB-L-cysteine was formed. A Vmax value of 0.0156±0.0008 units mg-1 and Km values of 150±18.3 mM for L-cysteine and 0.0579±0.0260 mM for DHB were obtained in this novel reaction. Furthermore, DhbE synthesized N-benzoyl-L-cysteine when benzoic acid and L-cysteine were used as substrates. Through the N-acylation reaction of DhbE, we also succeeded in the synthesis of N-aromatic acyl compounds that have never previously been reported to be produced by this enzymatic method.

Development and evaluation of FSSG: frequency scale for the symptoms of GERD.

BACKGROUND: The aim of this study was to produce a simplified questionnaire for evaluation of the symptoms of gastroesophageal reflux disease (GERD). METHODS: A total of 124 patients with an endoscopic diagnosis of GERD completed a 50-part questionnaire, requiring only "yes" or "no" answers, that covered various symptoms related to the upper gastrointestinal tract, as well as psychosomatic symptoms. The 12 questions to which patients most often answered "yes" were selected, and were assigned scores (never = 0; occasionally = 1; sometimes = 2; often = 3; and always = 4) to produce a frequency scale for symptoms of GERD (FSSG). Sensitivity, specificity, and accuracy of the FSSG questionnaire were evaluated in another group of patients with GERD and non-GERD. The usefulness of this questionnaire was evaluated in 26 other GERD patients who were treated with proton pump inhibitors for 8 weeks. RESULTS: When the cutoff score was set at 8 points, the FSSG showed a sensitivity of 62%, a specificity of 59%, and an accuracy of 60%, whereas a cutoff score of 10 points altered these values to 55%, 69%, and 63%. The score obtained using the questionnaire correlated well with the extent of endoscopic improvement in patients with mild or severe GERD. CONCLUSIONS: This new questionnaire is useful for the objective evaluation of symptoms in GERD patients.

Proton pump inhibitors improve acid-related dyspepsia in gastroesophageal reflux disease patients.

It has been reported that proton pump inhibitors are more effective than H2 receptor antagonists in patients with functional dyspepsia. Dyspeptic symptoms that respond to proton pump inhibitors are classified as acid-related dyspepsia. A new questionnaire for assessing gastroesophageal reflux disease (GERD), the Frequency Scale for Symptoms of GERD, covers the 12 most common symptoms of GERD patients. A quantitative assessment of the changes of reflux symptoms and acid-related dyspepsia was made in GERD patients receiving proton pump inhibitor therapy. Sixty-eight GERD patients receiving proton pump inhibitor therapy completed the questionnaire before and after treatment for 8 weeks. There is a significant positive correlation between reflux symptoms and acid-related dyspepsia before and after therapy (r = 0.569 and r = 0.569; both P's < 0.001) and acid-related dyspepsia in patients with both nonerosive and erosive GERD. We conclude that GERD patients suffer not only from reflux symptoms, but also from acid-related dyspepsia, and proton pump inhibitors improve both types of symptoms.

Sp1 is a co-activator with Ets-1, and Net is an important repressor of the transcription of CTP:phosphocholine cytidylyltransferase alpha.

Phosphatidylcholine biosynthesis via the CDP-choline pathway is primarily regulated by CTP:phosphocholine cytidylyltransferase (CT) encoded by the Pcyt1a and Pcyt1b genes. Previously, we identified an Ets-1-binding site located at -49/-47 in the promoter of Pcyt1a as an important transcriptional element involved in basal CTalpha transcription (Sugimoto, H., Sugimoto, S., Tatei, K., Obinata, H., Bakovic, M., Izumi, T., and Vance, D. E. (2003) J. Biol. Chem. 278, 19716-19722). In this study, we determined whether or not there were other important elements and binding proteins for basal CTalpha transcription in the Pcyt1a promoter, and if other Ets family proteins bind to the Ets-1-binding site. The results indicate the formation of a ternary complex with Ets-1 binding at -49/-47 and Sp1 binding at -58/-54 of the Pcyt1a promoter that is important for activating CTalpha transcription. When nuclear extracts of COS-7 cells expressing various Ets family repressors were incubated with DNA probes, binding of Net to the probes was observed. Net dose-dependently depressed the promoter-luciferase activity by 98%, even when co-expressed with Ets-1. RNA interference targeting Net caused an increase of endogenous CTalpha mRNA. After synchronizing the cell cycle in NIH3T3 cells, CTalpha mRNA increased at the S-M phase corresponding to an increase of Ets-1 mRNA and a decrease of Net mRNA. These results indicated that Net is an important endogenous repressor for CTalpha transcription.

Postprandial water intake inhibits gastric antral motility with increase of cholecystokinin in humans.

OBJECTIVE: The effects of postprandial water intake on the gastrointestinal tract have not been systematically investigated in humans. MATERIAL AND METHODS: In 8 healthy volunteers, the gastric antral pressure was measured with a strain gauge transducer, while the esophageal and lower esophageal sphincter pressures were measured with an infused catheter with a Dent sleeve. The esophageal pH at 5 cm above the lower sphincter was measured with a microglass electrode. A standard test meal (560 kcal) was eaten and 500 ml water was ingested 1 h later. The plasma cholecystokinin level was assessed at 4-min intervals. As a control, the same study was done on another day with sham water intake. RESULTS: At 4 min after water intake, there was a significant decrease in gastric antral motility and a significant increase in the plasma cholecystokinin level. Water intake also significantly increased the occurrence of gastroesophageal reflux. CONCLUSIONS: The rapid increase in cholecystokinin after water intake may be initiated by a feedback mechanism related to inflow of fatty chyme into the duodenum that inhibits gastric antral activity.

Diagnosis of gastroesophageal reflux disease using a new questionnaire.

BACKGROUND AND AIM: An early and accurate evaluation by a general practitioner is needed to screen out non-gastroesophageal reflux disease (GERD) patients. A recent questionnaire (QUEST) highlighted problems with specificity and complexity, so the aim of the present study was to design a simplified questionnaire. METHODS: When admitted to hospital to undergo an upper gastrointestinal endoscopy for suspected GERD, 333 patients completed a 50-item questionnaire requiring 'yes/no' answers to different combinations of questions relating to symptoms of upper gastrointestinal tract conditions (e.g. GERD, ulcers and functional dyspepsia) and psychosomatic symptoms. The endoscopic diagnosis was then correlated with the rate of positive answers to each question. RESULTS: Based on the analysis of the 50 items, the 8-10 questions most often answered affirmatively by each of the GERD and non-GERD groups were chosen for the simplified questionnaire. Three draft questionnaires were compiled. After calculating the sensitivity, specificity and accuracy in relation to the diagnosis of GERD and other conditions, it was found that questionnaire B (selection of persons answering 'yes' to at least one of questions 1-5 and exclusion of persons answering 'yes' to at least three of questions 7-10) had a high sensitivity, high specificity and low false positive rate. CONCLUSION: A novel questionnaire was developed. It was designed to detect the symptoms of GERD while simultaneously excluding non-GERD patients. This simplified nine-item simplified questionnaire had a sensitivity of 79.8%, a specificity of 53.6% and an accuracy of 63.4%.

Identification of Ets-1 as an important transcriptional activator of CTP:phosphocholine cytidylyltransferase alpha in COS-7 cells and co-activation with transcriptional enhancer factor-4.

Phosphatidylcholine biosynthesis via the CDP-choline pathway is primarily regulated by CTP:phosphocholine cytidylyltransferase (CT). Transcriptional enhancer factor-4 (TEF-4) enhances the transcription of CTalpha in COS-7 cells by interactions with the basal transcription machinery (Sugimoto, H., Bakovic, M., Yamashita, S., and Vance, D.E. (2001) J. Biol. Chem. 276,12338-12344). To identify the most important transcription factor involved in basal CTalpha transcription, we made CTalpha promoter-deletion and -mutated constructs linked to a luciferase reporter and transfected them into COS-7 cells. The results indicate that an important site regulating basal CTalpha transcription is -53/-47 (GACTTCC), which is a putative consensus-binding site of Ets transcription factors (GGAA) in the opposite orientation. Gel shift analyses indicated the existence of a binding protein for -53/-47 (GACTTCC) in nuclear extracts of COS-7 cells. When anti-Ets-1 antibody was incubated with the probe in gel shift analyses, the intensity of the binding protein was decreased. The binding of endogenous Ets-1 to the promoter probe was increased when TEF-4 was expressed; however, the amount of Ets-1 detected by immunoblotting was unchanged. When cells were transfected with Ets-1 cDNA, the luciferase activity of CTalpha promoter constructs was greatly enhanced. Co-transfection experiments with Ets-1 and TEF-4 showed enhanced expression of reporter constructs as well as CTalpha mRNA. These results suggest that Ets-1 is an important transcriptional activator of the CTalpha gene and that Ets-1 activity is enhanced by TEF-4.